The overall goal of this proposal is to use the RAS-RAF-MEK-ERK (MAPK/ERK) signaling pathway as a model to develop small molecule inhibitors that specifically target kinase dimers as lead compounds for therapeutic development. The MAPK/ERK signaling pathway is frequently activated in human cancer and the activating BRAF V600E mutant (BRAFV600E) in particular accounts for about 7% of all cancers and about 50% of malignant melanoma tumors, thus highlighting BRAF and MAPK/ERK signaling as important targets for therapy. Although many melanoma patients treated with highly selective BRAFV600E mutant inhibitors (BRAFi), downstream MEK inhibitors (MEKi) or BRAFi/MEKi combinations, initially respond, nearly all develop drug resistance and progress at a median of about 1 year survival for combination therapy. Intrinsic or acquired resistance to BRAF inhibitors often arise through potent activation or reactivation of the MAPK pathway, typically through mutation of upstream RAS or downstream MEK. Biochemically, MAPK activation is driven by RAF dimerization, whereby drug-bound BRAF or BRAFV600E mediates drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called ?transactivation? or ?paradoxical activation.? To test if transactivation could be overcome by selectively targeting RAF dimers, we used BRAFV600E and vemurafenib as a model system to develop bivalent kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We showed that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells, but promotes an inactive dimeric BRAFV600E conformation that is unable to undergo transactivation. In related studies, we also chemically linked vemurafenib to the MEK inhibitor G894, and demonstrated that this compound inhibited growth of BRAFi-resistant cells more effectively than either vemurafenib or G894 alone or a vemurafenib/G894 combination. We have also carried out a high-throughput assay to measure inhibition of the BRAF/MEK interaction with the goal of developing small molecule BRAF- MEK dimerizatioin inhibitors. With this preliminary data on hand, we will now (1) Develop inhibitors that target BRAF homo- and hetero-dimers with BRAFV600E, BRAF or CRAF, and (2) Develop inhibitors that target RAF/MEK complexes. These studies will lead to the development of small molecule inhibitors to target BRAF homo- and hetero-dimers with BRAF, CRAF, BRAFV600E and MEK that we anticipate will be superior to inhibitors that target kinase monomers for inhibiting MAPK/ERK signaling in melanoma and melanoma resistant to therapy. These lead inhibitors will provide a new paradigm for the development of a new family of melanoma drugs.